CN103005157A - Novel efficient synbiotics intestines-targeting microspheric capsule and preparation method thereof - Google Patents
Novel efficient synbiotics intestines-targeting microspheric capsule and preparation method thereof Download PDFInfo
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- CN103005157A CN103005157A CN201210539339XA CN201210539339A CN103005157A CN 103005157 A CN103005157 A CN 103005157A CN 201210539339X A CN201210539339X A CN 201210539339XA CN 201210539339 A CN201210539339 A CN 201210539339A CN 103005157 A CN103005157 A CN 103005157A
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- 235000019722 synbiotics Nutrition 0.000 title claims abstract description 31
- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- 239000002775 capsule Substances 0.000 title abstract description 8
- 239000004005 microsphere Substances 0.000 title abstract 5
- 239000003094 microcapsule Substances 0.000 claims abstract description 22
- 229920000392 Zymosan Polymers 0.000 claims abstract description 16
- 239000000203 mixture Substances 0.000 claims abstract description 14
- 235000013406 prebiotics Nutrition 0.000 claims abstract description 14
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 50
- 241000894006 Bacteria Species 0.000 claims description 40
- 239000001963 growth medium Substances 0.000 claims description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 28
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 27
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 27
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 25
- 241000193171 Clostridium butyricum Species 0.000 claims description 25
- 239000004310 lactic acid Substances 0.000 claims description 25
- 235000014655 lactic acid Nutrition 0.000 claims description 25
- 238000000855 fermentation Methods 0.000 claims description 24
- 230000004151 fermentation Effects 0.000 claims description 24
- 150000004676 glycans Chemical class 0.000 claims description 19
- 229920001282 polysaccharide Polymers 0.000 claims description 19
- 239000005017 polysaccharide Substances 0.000 claims description 19
- 239000012530 fluid Substances 0.000 claims description 18
- 239000006228 supernatant Substances 0.000 claims description 18
- 238000004458 analytical method Methods 0.000 claims description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- 239000012153 distilled water Substances 0.000 claims description 12
- 244000005700 microbiome Species 0.000 claims description 12
- 239000000843 powder Substances 0.000 claims description 12
- 230000001954 sterilising effect Effects 0.000 claims description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 238000004108 freeze drying Methods 0.000 claims description 9
- 238000001556 precipitation Methods 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 9
- 238000005303 weighing Methods 0.000 claims description 9
- 230000001580 bacterial effect Effects 0.000 claims description 7
- 238000012360 testing method Methods 0.000 claims description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 6
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- 238000000576 coating method Methods 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 238000011081 inoculation Methods 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
- 239000003223 protective agent Substances 0.000 claims description 6
- AKEJUJNQAAGONA-UHFFFAOYSA-N sulfur trioxide Chemical compound O=S(=O)=O AKEJUJNQAAGONA-UHFFFAOYSA-N 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 5
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 4
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 4
- 230000004048 modification Effects 0.000 claims description 4
- 238000012986 modification Methods 0.000 claims description 4
- 239000000243 solution Substances 0.000 claims description 4
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 3
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 3
- 108010010803 Gelatin Proteins 0.000 claims description 3
- 229920002472 Starch Polymers 0.000 claims description 3
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 3
- 239000000853 adhesive Substances 0.000 claims description 3
- 230000001070 adhesive effect Effects 0.000 claims description 3
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 3
- 235000015278 beef Nutrition 0.000 claims description 3
- 239000012267 brine Substances 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 235000020247 cow milk Nutrition 0.000 claims description 3
- 238000000502 dialysis Methods 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 238000012869 ethanol precipitation Methods 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 229920000159 gelatin Polymers 0.000 claims description 3
- 239000008273 gelatin Substances 0.000 claims description 3
- 235000019322 gelatine Nutrition 0.000 claims description 3
- 235000011852 gelatine desserts Nutrition 0.000 claims description 3
- 238000005469 granulation Methods 0.000 claims description 3
- 230000003179 granulation Effects 0.000 claims description 3
- 238000000703 high-speed centrifugation Methods 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 230000001376 precipitating effect Effects 0.000 claims description 3
- 239000000376 reactant Substances 0.000 claims description 3
- 150000003839 salts Chemical class 0.000 claims description 3
- 239000002002 slurry Substances 0.000 claims description 3
- 235000010413 sodium alginate Nutrition 0.000 claims description 3
- 229940005550 sodium alginate Drugs 0.000 claims description 3
- 239000000661 sodium alginate Substances 0.000 claims description 3
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 claims description 3
- 239000007921 spray Substances 0.000 claims description 3
- 238000005507 spraying Methods 0.000 claims description 3
- 239000008107 starch Substances 0.000 claims description 3
- 235000019698 starch Nutrition 0.000 claims description 3
- 238000003828 vacuum filtration Methods 0.000 claims description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 2
- 241001465754 Metazoa Species 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 7
- 235000018291 probiotics Nutrition 0.000 abstract description 7
- 239000006041 probiotic Substances 0.000 abstract description 5
- 238000000034 method Methods 0.000 abstract description 4
- 230000036039 immunity Effects 0.000 abstract description 3
- 239000000654 additive Substances 0.000 abstract description 2
- 230000000996 additive effect Effects 0.000 abstract description 2
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 230000007613 environmental effect Effects 0.000 abstract description 2
- 230000036541 health Effects 0.000 abstract description 2
- 241001112696 Clostridia Species 0.000 abstract 1
- 241001508691 Martes zibellina Species 0.000 abstract 1
- 229940066544 lactobacillus sporogenes Drugs 0.000 abstract 1
- 230000001737 promoting effect Effects 0.000 abstract 1
- 231100000331 toxic Toxicity 0.000 abstract 1
- 230000002588 toxic effect Effects 0.000 abstract 1
- 230000002496 gastric effect Effects 0.000 description 6
- 230000004083 survival effect Effects 0.000 description 5
- 230000003115 biocidal effect Effects 0.000 description 4
- 239000003814 drug Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
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- 229940088710 antibiotic agent Drugs 0.000 description 2
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- 231100000957 no side effect Toxicity 0.000 description 2
- 238000004088 simulation Methods 0.000 description 2
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- 208000031295 Animal disease Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000002155 anti-virotic effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
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- 210000004051 gastric juice Anatomy 0.000 description 1
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- 230000000968 intestinal effect Effects 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
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- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 150000003222 pyridines Chemical class 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- UDYFLDICVHJSOY-UHFFFAOYSA-N sulfur trioxide pyridine complex Chemical compound O=S(=O)=O.C1=CC=NC=C1 UDYFLDICVHJSOY-UHFFFAOYSA-N 0.000 description 1
- 241001148471 unidentified anaerobic bacterium Species 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a novel efficient synbiotics intestines-targeting microspheric capsule and a preparation method of the novel efficient synbiotics intestines-targeting microspheric capsule. The novel efficient synbiotics intestines-targeting microspheric capsule comprises 30-40% of probiotics and 60-70% of prebiotics, wherein the probiotics is a mixture composed of 40 to 50% of clostridia butyricum and 50 to 60% of lactobacillus sporogenes; and the prebiotics is the mixture composed of 60 to 70% of cerevisiae zymosan and 30 to 40% of sulphated cerevisiae zymosan. The novel micro-capsule synbiotics additive for the feed, prepared by the method disclosed by the invention, plays the effects of both the probiotics and the prebiotics, has remarkable effects of improving the utilization rate of the feed, promoting the growth of animal and improving the resistance and immunity of the human body; and the novel efficient synbiotics intestines-targeting microspheric capsule is sable in quality, safe, and free from toxic and side effect, brings no residues and pollution, and is beneficial for the human health and environmental protection.
Description
Technical field
The present invention relates to a kind of feed addictive, be specifically related to a kind of new and effective colon-targeted microcapsule of synbiotic and preparation method thereof.
Background technology
Antibiotic is being brought into play great function for a long time always in animal produces, it can treat and prevent Animal diseases to occur effectively, but antibiotic lasting use can cause the problems such as bacterial drug resistance and animal product medicament residue, therefore seek the feed addictive substitute antibiotics feed addictive with developing green, pollution-free, noresidue, become the focus of present feed industry research.
Synbiotics (synbiotics) claims again symphysis unit, by Gibson(1995) at first propose.It refers to probio (Probiotics) and the biologic product that prebiotics (Prebiotics) is combined with, and is characterized in bringing into play simultaneously the effect of probio and prebiotics.The result of use of Synbiotics is equivalent to or is better than antibiotic, and its cost is lower than the antibacterials such as antibiotic, and does not have the problem of medicament residue.Synbiotics (synbiotics) both can have been brought into play the physiological property of probio as microecologic regulator, optionally increased again probio quantity, made the effect of probio more remarkable lasting.It is the main direction of microecologic regulator Future Development.At present, there is following problem in most of little ecological live bacteria agents: viable bacteria content is low in the probiotics preparation, and production cost is high; Probio is anaerobic bacteria, is subject to the external environment factor affecting, and shelf life is short under the normal temperature, and can advance the low problem of HE probio survival rate through gastric juice.
Summary of the invention
The purpose of this invention is to provide a kind of new and effective colon-targeted microcapsule of synbiotic and preparation method thereof, improve the probio survival rate, prolong the shelf life of probio in the Synbiotics, good stability, safely, have no side effect.
The technical solution used in the present invention is as follows:
A kind of new and effective colon-targeted microcapsule of synbiotic, its component is: the probio of 30-40% and the prebiotics of 60-70%; The mixture that described probio is comprised of the lactic acid bacillus of the clostridium butyricum of 40-50% and 50-60%; The mixture that described prebiotics is comprised of the sulphation Analysis of Polysaccharide From Saccharomyces Cerevisiae of the Analysis of Polysaccharide From Saccharomyces Cerevisiae of 60-70% and 30-40%.
The preparation method of new and effective colon-targeted microcapsule of synbiotic may further comprise the steps:
(1) preparation of probio
1. the preparation of lactic acid bacillus
The lactic acid bacillus culture medium is: yeast extract 10g, peptone 10g, glucose 6g, MgSO
47H
2O 1g, MnSO
40.05g, K
2HPO
42g, distilled water 1L, PH=7.0-7.2, sterilising conditions are 120-125 ℃, 20-30min;
The first step: get that bacterial classification inoculation enters to be equipped with in the test tube of lactic acid bacillus culture medium on the inclined-plane, 35-38 ℃ of constant temperature leaves standstill anaerobism cultivates 20-24h, obtains the first order seed bacterium, takes out and puts into Refrigerator store; Stand-by;
Second step: the ratio in the 3-5% of lactic acid bacillus culture medium is inoculated into the first order seed bacterium in the lactic acid bacillus culture medium, obtains the secondary seed bacterium;
The 3rd step: the component ratio by the lactic acid bacillus culture medium takes by weighing each composition, pour in the microorganism fermentation tank, add water to 20L, in 110-120 ℃ of lower high-temp steam sterilizing 20-30min, treat that it is cooled to 35-40 ℃, add the secondary seed bacterium in the ratio of the 5-10% of lactic acid bacillus kind culture volume, stir, carry out fermented and cultured, fermentation temperature 35-45 ℃, fermentation time 20-30h;
The 4th step: in microorganism fermentation tank, take out zymotic fluid, after it is cooled to normal temperature, centrifugal 15-20min removes supernatant under the 2500-3000r/min rotating speed, adds the 10-20g trehalose in every kilogram of wet thallus as protective agent, adding 200-300g macropore starch adsorbs again, stir, at-35～-40 ℃ of lower pre-freeze 1-2h, dry 25-30h under vacuum 10.00-12.00Pa condition then, pulverize grind into powder, for subsequent use;
2. the preparation of clostridium butyricum
The clostridium butyricum culture medium is: yeast extract 5g, peptone 10g, glucose 10g, beef extract 3g, ammonium sulfate 1g, NaCl2g, dipotassium hydrogen phosphate 4g, sodium acid carbonate 1g, MnSO
40.2g%, MgSO
47H
2O 0.5g, CaCO
31g, distilled water 1L, PH=6.8-7.0, sterilising conditions are 120-130 ℃, 20-30min;
The first step: get that bacterial classification inoculation enters to be equipped with in the test tube of clostridium butyricum culture medium on the inclined-plane, 36-38 ℃ of constant temperature leaves standstill anaerobism cultivates 20-24h, obtains the first order seed bacterium, takes out and puts into Refrigerator store; Stand-by;
Second step: the ratio in the 4-6% of clostridium butyricum culture medium is inoculated into the first order seed bacterium in the clostridium butyricum culture medium, obtains the secondary seed bacterium;
The 3rd step: the component ratio by the clostridium butyricum culture medium takes by weighing each composition, pour in the microorganism fermentation tank, add water to 22L, in 110-120 ℃ of lower high-temp steam sterilizing 20-30min, treat that it is cooled to 35-40 ℃, add the secondary seed bacterium in the ratio of the 8-12% of clostridium butyricum kind culture volume, stir, carry out fermented and cultured, fermentation temperature 35-45 ℃, fermentation time 32-40h;
The 4th step: in microorganism fermentation tank, take out zymotic fluid, after it is cooled to normal temperature, centrifugal 15-20min under the 2500-3000r/min rotating speed, remove supernatant, bacterium mud is washed out as protective agent with the cow's milk juice of 20-30%, then it is mixed with the coating buffer that contains gelatin, homogeneous is coated, freeze drying, grind into powder, for subsequent use;
3. the extraction of Analysis of Polysaccharide From Saccharomyces Cerevisiae
First beer yeast slurry is carried out preliminary treatment: clean with distilled water, vacuum filtration carries out the washing second time again, behind the placement 0.5-1 h, outwell supernatant, centrifugal, 0.5% sodium acid carbonate that adds twice yeast paste volume washs 1-2 h, wash again twice, centrifugal, abandon supernatant and obtain clean yeast.Be converted to dry ferment weight according to its water content, adding an amount of water and salt makes yeast and brine concentration be 8-10%, solution is placed high pressure homogenizer, homogeneous is 2-3 time under temperature 110-120 ℃, the condition of high pressure 60-70MPa, cooling, high speed centrifugation, keep supernatant, be concentrated into the 1/4-1/5 of original volume,: make precipitating reagent with 95% ethanol, precipitation 10-12h, the volume ratio of ethanol and supernatant is 2-3:1, precipitation is washed 2 times with acetone, with ether washing 1 time, will be deposited at last 30-40 ℃ of lower dry 12-16h, namely get Analysis of Polysaccharide From Saccharomyces Cerevisiae;
4. sulphation modification zymosan
2.0-4.0:1 takes by weighing sulfur trioxide and Analysis of Polysaccharide From Saccharomyces Cerevisiae in reactor in mass ratio, add the dissolving of 20-30 mL pyridine, start mixer, after the dissolving, at 60-70 ℃ of water-bath 2-3h, reaction is cooled to room temperature after finishing, reactant liquor is invaded in the distilled water, regulate the pH value to 7-8 with 2.5 mol/L NaOH, the centrifugal insoluble matter of abandoning, the 95% ethanol precipitation polysaccharide that adds 4-5 times of volume is separated out precipitation, to precipitate water-soluble, with distill water dialysis 36-48 h, concentrated, freeze drying gets the sulphation zymosan;
5. the preparation of colon-targeted microcapsule of synbiotic
Adopt fluid bed coating method: the dry bacterium powder after freeze drying is mixed with zymosan, sulphation zymosan, then mixed dry powder is added in the fluid bed, heat by the hot-air that moves upward, material keeps good suspended state, select sodium alginate to spray into by spraying system as adhesive and carry out the micro-capsule granulation, airtight package keeps in Dark Place.
Beneficial effect of the present invention:
The present invention adopts the broken yeast cell wall of high pressure homogenization method to extract zymosan, simple process, and raw material is cheap to be easy to get, and production cost is low, and environmental pollution is less, is suitable for suitability for industrialized production; After adopting sulfur trioxide-pyridine method that zymosan is carried out sulphation modification, strengthened the biologically active of original polysaccharide, improve efficiency of feed utilization, promote growth of animal, improve the animal resistance, the resistance and the immunity that strengthen body have remarkable effect, and also pathogen and strengthened the animal body anti-virus ability in the establishment animal body solves the proactive problem of long-term puzzlement livestock and poultry industry virus disease.The novel micro-capsule Synbiotic feed that the present invention produces is brought into play the double action of probio and prebiotics simultaneously with additive; has the raising efficiency of feed utilization; promote growth of animal; strengthen the resistance of body and the remarkable effect of immunity; constant product quality, safely, have no side effect noresidue; pollution-free, be conducive to health of human body and environmental protection.
The specific embodiment
Embodiment 1: the preparation of colon-targeted microcapsule of synbiotic
A kind of new and effective colon-targeted microcapsule of synbiotic, its component is: 40% probio and 60% prebiotics; The mixture that described probio is comprised of the lactic acid bacillus of 50% clostridium butyricum and 50%; The mixture that described prebiotics is comprised of the sulphation Analysis of Polysaccharide From Saccharomyces Cerevisiae of 70% Analysis of Polysaccharide From Saccharomyces Cerevisiae and 30%.
The preparation method of new and effective colon-targeted microcapsule of synbiotic may further comprise the steps:
(1) preparation of probio
1. the preparation of lactic acid bacillus
The lactic acid bacillus culture medium is: yeast extract 10g, peptone 10g, glucose 6g, MgSO
47H
2O 1g, MnSO
40.05g, K
2HPO
42g, distilled water 1L, PH=7.2, sterilising conditions are 121 ℃, 30min;
The first step: get that bacterial classification inoculation enters to be equipped with in the test tube of lactic acid bacillus culture medium on the inclined-plane, 38 ℃ of constant temperature leave standstill anaerobism cultivates 22h, obtains the first order seed bacterium, takes out and puts into Refrigerator store; Stand-by;
Second step: 4% ratio in the lactic acid bacillus culture medium is inoculated into the first order seed bacterium in the lactic acid bacillus culture medium, obtains the secondary seed bacterium;
The 3rd step: the component ratio by the lactic acid bacillus culture medium takes by weighing each composition, pour in the microorganism fermentation tank, add water to 20L, in 115 ℃ of lower high-temp steam sterilizing 30min, treat that it is cooled to 35 ℃, add the secondary seed bacterium in 8% ratio of lactic acid bacillus kind culture volume, stir, carry out fermented and cultured, 40 ℃ of fermentation temperatures, fermentation time 25h;
The 4th step: in microorganism fermentation tank, take out zymotic fluid, after it is cooled to normal temperature, centrifugal 15min removes supernatant under the 3000r/min rotating speed, adds the 12g trehalose in every kilogram of wet thallus as protective agent, adding 240g macropore starch adsorbs again, stir, at-35 ℃ of lower pre-freeze 1.5h, dry 30h under vacuum 10.26Pa condition then, pulverize grind into powder, for subsequent use;
2. the preparation of clostridium butyricum
The clostridium butyricum culture medium is: yeast extract 5g, peptone 10g, glucose 10g, beef extract 3g, ammonium sulfate 1g, NaCl2g, dipotassium hydrogen phosphate 4g, sodium acid carbonate 1g, MnSO
40.2g%, MgSO
47H
2O 0.5g, CaCO
31g, distilled water 1L, PH=7.0, sterilising conditions are 124 ℃, 30min;
The first step: get that bacterial classification inoculation enters to be equipped with in the test tube of clostridium butyricum culture medium on the inclined-plane, 36 ℃ of constant temperature leave standstill anaerobism cultivates 24h, obtains the first order seed bacterium, takes out and puts into Refrigerator store; Stand-by;
Second step: 5% ratio in the clostridium butyricum culture medium is inoculated into the first order seed bacterium in the clostridium butyricum culture medium, obtains the secondary seed bacterium;
The 3rd step: the component ratio by the clostridium butyricum culture medium takes by weighing each composition, pour in the microorganism fermentation tank, add water to 22L, in 120 ℃ of lower high-temp steam sterilizing 20min, treat that it is cooled to 40 ℃, add the secondary seed bacterium in 10% ratio of clostridium butyricum kind culture volume, stir, carry out fermented and cultured, 42 ℃ of fermentation temperatures, fermentation time 38h;
The 4th step: in microorganism fermentation tank, take out zymotic fluid, after it is cooled to normal temperature, centrifugal 20min under the 2500r/min rotating speed, remove supernatant, the cow's milk juice with 25% washes out bacterium mud as protective agent, then it is mixed with the coating buffer that contains gelatin, homogeneous is coated, freeze drying, grind into powder, for subsequent use;
3. the extraction of Analysis of Polysaccharide From Saccharomyces Cerevisiae
First beer yeast slurry is carried out preliminary treatment: clean with distilled water, vacuum filtration carries out second time again and washes, place 1 h after, outwell supernatant, centrifugal, 0.5% sodium acid carbonate that adds twice yeast paste volume washs 2 h, wash again twice, centrifugal, abandon supernatant and obtain clean yeast.Be converted to dry ferment weight according to its water content, adding an amount of water and salt makes yeast and brine concentration be 8%, solution is placed high pressure homogenizer, homogeneous is 3 times under the condition of 120 ℃ of temperature, high pressure 65MPa, cooling, high speed centrifugation, keep supernatant, be concentrated into 1/5 of original volume,: make precipitating reagent with 95% ethanol, precipitation 12h, the volume ratio of ethanol and supernatant is 3:1, precipitation is washed 2 times with acetone, with ether washing 1 time, will be deposited at last 35 ℃ of lower dry 15h, namely get Analysis of Polysaccharide From Saccharomyces Cerevisiae;
4. sulphation modification zymosan
3.0:1 takes by weighing sulfur trioxide and Analysis of Polysaccharide From Saccharomyces Cerevisiae in reactor in mass ratio, adds the dissolving of 25 mL pyridines, starts mixer, after the dissolving, at 60 ℃ of water-bath 3h, reaction is cooled to room temperature after finishing, reactant liquor is invaded in the distilled water, regulate pH value to 7.5, the centrifugal insoluble matter of abandoning with 2.5 mol/L NaOH, the 95% ethanol precipitation polysaccharide that adds 4 times of volumes is separated out precipitation, to precipitate water-soluble, with distill water dialysis 42 h, concentrated, freeze drying gets the sulphation zymosan;
5. the preparation of colon-targeted microcapsule of synbiotic
Adopt fluid bed coating method: the dry bacterium powder after freeze drying is mixed with zymosan, sulphation zymosan, then mixed dry powder is added in the fluid bed, heat by the hot-air that moves upward, material keeps good suspended state, select sodium alginate to spray into by spraying system as adhesive and carry out the micro-capsule granulation, airtight package keeps in Dark Place.
Embodiment 2: colon-targeted microcapsule of synbiotic test storage period
The colon-targeted microcapsule of synbiotic that embodiment 1 is made is packed in the medicine bottle, sealing is waxed, respectively at 4 ℃ of refrigerators, 25 ℃ of room temperatures, 37 ℃ of acceleration experiments and the lower storage at a constant temperature of relative humidity 75% (RH75%), measure respectively under the different time in the capsule total bacteria count and store after 1 year the content of 3 kinds of bacteriums in the capsule, the results are shown in Table 1,2.
Table 1 colon-targeted microcapsule of synbiotic viable count under different temperatures changes
Table 2 is stored after 1 year the content of 2 kinds of bacteriums in the colon-targeted microcapsule of synbiotic under different temperatures
By table 1 and table 2 as can be known, two kinds of bacteriums stored survival rate through 1 year and are respectively under different temperatures: only be 61.5% when being 77.4%, 37 ℃ when being 87.8%, 25 ℃ in the time of 4 ℃; Being 1.12:1 when 4 ℃ of lactic acid bacillus, clostridium butyricum two all bacterium viable bacteria ratios, is 1.11:1 in the time of 25 ℃, is 1:2.1 in the time of 37 ℃.
Embodiment 3: simulation simulated gastric fluid and intestinal juice experiment
Get 4 capsules, respectively getting 1 puts in the 250mL conical flask that fills 100mL simulation simulated gastric fluid, it is the 150r/min concussion dissolving on 37 ℃ the thermostatic water bath vibrator in temperature, in 4h, take a sample once every 0.5h, in and acidity, the simulated gastric fluid of quick mixing of learning from else's experience detects its survival rate and viable count, gets average to analyze dissolving situation in simulated gastric fluid, the results are shown in Table 3.
Table 3 Synbiotics is the dissolving situation in simulated gastric fluid
As shown in Table 3, in simulated gastric fluid, the viable bacteria survival rate reaches 78.2% in 2h, and total viable bacteria is 1.76x10
10Cfug
-1Survival rate descends slowly from 1.5h, is because in 37 ℃ of lower prebioticses and probio coupling, produces certain coordinative role and has strengthened the probio competitive advantage.
Claims (2)
1. a new and effective colon-targeted microcapsule of synbiotic is characterized in that, its component is: the probio of 30-40% and the prebiotics of 60-70%; The mixture that described probio is comprised of the lactic acid bacillus of the clostridium butyricum of 40-50% and 50-60%; The mixture that described prebiotics is comprised of the sulphation Analysis of Polysaccharide From Saccharomyces Cerevisiae of the Analysis of Polysaccharide From Saccharomyces Cerevisiae of 60-70% and 30-40%.
2. the preparation method of a new and effective colon-targeted microcapsule of synbiotic as claimed in claim 1 is characterized in that may further comprise the steps:
(1) preparation of probio
1. the preparation of lactic acid bacillus
The lactic acid bacillus culture medium is: yeast extract 10g, peptone 10g, glucose 6g, MgSO
47H
2O 1g, MnSO
40.05g, K
2HPO
42g, distilled water 1L, PH=7.0-7.2, sterilising conditions are 120-125 ℃, 20-30min;
The first step: get that bacterial classification inoculation enters to be equipped with in the test tube of lactic acid bacillus culture medium on the inclined-plane, 35-38 ℃ of constant temperature leaves standstill anaerobism cultivates 20-24h, obtains the first order seed bacterium, takes out and puts into Refrigerator store; Stand-by;
Second step: the ratio in the 3-5% of lactic acid bacillus culture medium is inoculated into the first order seed bacterium in the lactic acid bacillus culture medium, obtains the secondary seed bacterium;
The 3rd step: the component ratio by the lactic acid bacillus culture medium takes by weighing each composition, pour in the microorganism fermentation tank, add water to 20L, in 110-120 ℃ of lower high-temp steam sterilizing 20-30min, treat that it is cooled to 35-40 ℃, add the secondary seed bacterium in the ratio of the 5-10% of lactic acid bacillus kind culture volume, stir, carry out fermented and cultured, fermentation temperature 35-45 ℃, fermentation time 20-30h;
The 4th step: in microorganism fermentation tank, take out zymotic fluid, after it is cooled to normal temperature, centrifugal 15-20min removes supernatant under the 2500-3000r/min rotating speed, adds the 10-20g trehalose in every kilogram of wet thallus as protective agent, adding 200-300g macropore starch adsorbs again, stir, at-35～-40 ℃ of lower pre-freeze 1-2h, dry 25-30h under vacuum 10.00-12.00Pa condition then, pulverize grind into powder, for subsequent use;
2. the preparation of clostridium butyricum
The clostridium butyricum culture medium is: yeast extract 5g, peptone 10g, glucose 10g, beef extract 3g, ammonium sulfate 1g, NaCl2g, dipotassium hydrogen phosphate 4g, sodium acid carbonate 1g, MnSO
40.2g%, MgSO
47H
2O 0.5g, CaCO
31g, distilled water 1L, PH=6.8-7.0, sterilising conditions are 120-130 ℃, 20-30min;
The first step: get that bacterial classification inoculation enters to be equipped with in the test tube of clostridium butyricum culture medium on the inclined-plane, 36-38 ℃ of constant temperature leaves standstill anaerobism cultivates 20-24h, obtains the first order seed bacterium, takes out and puts into Refrigerator store; Stand-by;
Second step: the ratio in the 4-6% of clostridium butyricum culture medium is inoculated into the first order seed bacterium in the clostridium butyricum culture medium, obtains the secondary seed bacterium;
The 3rd step: the component ratio by the clostridium butyricum culture medium takes by weighing each composition, pour in the microorganism fermentation tank, add water to 22L, in 110-120 ℃ of lower high-temp steam sterilizing 20-30min, treat that it is cooled to 35-40 ℃, add the secondary seed bacterium in the ratio of the 8-12% of clostridium butyricum kind culture volume, stir, carry out fermented and cultured, fermentation temperature 35-45 ℃, fermentation time 32-40h;
The 4th step: in microorganism fermentation tank, take out zymotic fluid, after it is cooled to normal temperature, centrifugal 15-20min under the 2500-3000r/min rotating speed, remove supernatant, bacterium mud is washed out as protective agent with the cow's milk juice of 20-30%, then it is mixed with the coating buffer that contains gelatin, homogeneous is coated, freeze drying, grind into powder, for subsequent use;
3. the extraction of Analysis of Polysaccharide From Saccharomyces Cerevisiae
First beer yeast slurry is carried out preliminary treatment: clean with distilled water, vacuum filtration carries out the washing second time again, behind the placement 0.5-1 h, outwell supernatant, centrifugal, 0.5% sodium acid carbonate that adds twice yeast paste volume washs 1-2 h, wash again twice, centrifugal, abandon supernatant and obtain clean yeast; Be converted to dry ferment weight according to its water content, adding an amount of water and salt makes yeast and brine concentration be 8-10%, solution is placed high pressure homogenizer, homogeneous is 2-3 time under temperature 110-120 ℃, the condition of high pressure 60-70MPa, cooling, high speed centrifugation, keep supernatant, be concentrated into the 1/4-1/5 of original volume,: make precipitating reagent with 95% ethanol, precipitation 10-12h, the volume ratio of ethanol and supernatant is 2-3:1, precipitation is washed 2 times with acetone, with ether washing 1 time, will be deposited at last 30-40 ℃ of lower dry 12-16h, namely get Analysis of Polysaccharide From Saccharomyces Cerevisiae;
4. sulphation modification zymosan
2.0-4.0:1 takes by weighing sulfur trioxide and Analysis of Polysaccharide From Saccharomyces Cerevisiae in reactor in mass ratio, add the dissolving of 20-30 mL pyridine, start mixer, after the dissolving, at 60-70 ℃ of water-bath 2-3h, reaction is cooled to room temperature after finishing, reactant liquor is invaded in the distilled water, regulate the pH value to 7-8 with 2.5 mol/L NaOH, the centrifugal insoluble matter of abandoning, the 95% ethanol precipitation polysaccharide that adds 4-5 times of volume is separated out precipitation, to precipitate water-soluble, with distill water dialysis 36-48 h, concentrated, freeze drying gets the sulphation zymosan;
5. the preparation of colon-targeted microcapsule of synbiotic
Adopt fluid bed coating method: the dry bacterium powder after freeze drying is mixed with zymosan, sulphation zymosan, then mixed dry powder is added in the fluid bed, heat by the hot-air that moves upward, material keeps good suspended state, select sodium alginate to spray into by spraying system as adhesive and carry out the micro-capsule granulation, airtight package keeps in Dark Place.
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